MiR-145 is upregulated in the retarded preimplantation embryos and modulates cholesterol levels in mice preimplantation embryos through targeting Abca1

Background Preimplantation embryonic lethality is a driver of female infertility. Certain microRNAs (miRNAs) have previously been demonstrated to play important roles in the regulation of embryogenesis. Methods Normally developing blastocysts and arrested embryos were collected from patients undergoing intracytoplasmic sperm injection (ICSI), and the expression of specific miRNAs therein was evaluated by qPCR. The overexpression of target molecule miR-145 in early mice embryos was achieved via oocyte microinjection, enabling the subsequent monitoring of how such overexpression impacted embryonic development. Bioinformatics approaches were utilized to identify putative miR-145 target mRNAs, and luciferase reporter assessments were implemented to confirm the ability of miR-145 to regulate Abca1 in HEK293T cells. The functional relationship between miR-145 and Abca1 in the mice’s embryonic development was then confirmed through rescue assays. Results Abnormally increased miR-145 expression was observed in patients’ arrested embryos, and the exogenous overexpression of this miRNA significantly suppressed mural blastocyst formation. Mechanistically, miR-145 was found to bind to the 3′-untranslated area of the Abca1 mRNA in HK293T cells, thus suppressing its expression and increasing embryonic cholesterol levels. In line with the importance of these cholesterol levels to embryogenesis, the upregulation of Abca1 was sufficient to rescue the observed change in cholesterol levels and the associated retardation of mice embryonic development that occurred in response to the overexpression of miR-145. Conclusion The regulatory dynamics of the miR-145/Abca1 axis play an important role in shaping normal embryonic development.


Background
Infertility affects 10-15% of couples and represents a growing clinical challenge worldwide [1]. The most common causes of infertility are ovulatory dysfunction, male factor infertility, and tubal disease. In addition, approximately 15% of infertile couples have "unexplained infertility" [2]. The emergence of assisted reproductive technology (ART) has proven to be of great value for Page 2 of 10 Jian et al. Reproductive Biology and Endocrinology (2022) 20:168 couples suffering from infertility. Current live birth rates per ART treatment cycle are considerably higher when compared with the early years of ART, but overall, success rates remain low [3]. High rates of developmental arrest partly account for the observed low success rates, as only about 50% of embryos develop to the blastocyst stage despite improvements in culture media, laboratory equipment, and techniques [4]. Recent research derived from both embryonic studies and ART believes that preimplantation embryonic lethality (PEL) is a reason for initial female infertility [5]. In women with PEL, ovarian follicle expansion and ovulation usually proceed. Yet, women nonetheless suffer from recurrent in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) failures owing to a lack of successful fertilization or early embryonic arrest [6].
Dramatic changes ensure that a series of pivotal biological events proceed, including oocyte activation and maternal-to-zygotic transition coordinated with zygotic gene activation, followed by the first cell-fate decision and lineage-specific differentiation [7]. A precise regulatory network must function adequately to support such a significant shift in a short period. Epigenetic information is essential in maintaining cell characteristics and controlling gene expression. Epigenetic modifications, like DNA methylation, histone modifications, and noncoding RNA, have been found to play an irreplaceable role in the development of an embryo into a new individual [8].
MicroRNAs (miRNAs) are tiny (~ 22 nucleotides) non-coding RNAs that lack coding capability yet can control the permanence and expression of specific target mRNAs through binding to complementary succession within their 3′-untranslated region (UTR) domains. A great deal of data divulges that miRNAs are among the most abundant regulatory factors within cells, with individual miRNAs having the capability to control the expression of many target genes and individual genes similarly being subject to the regulatory activity of multiple miRNAs in some cases [9][10][11][12][13]. Both physiological and pathological activities have been attributed to miRNAs in human reproduction, and the dysregulated expression of many miRNAs has been noted in individuals suffering from specific reproductive disorders [14][15][16]. As such, there has been growing research interest in identifying and functionally characterizing those miRNAs influencing oocyte maturation and preimplantation embryonic development. For instance, Byrne et al. [17] proved that the loss of the miR-290-295 cluster in mice was associated with embryonic lethality due to the dysregulation of genes related to early embryonic development [18]. Consistently, the upregulation or downregulation of particular sets of miRNAs has been noted in many species in embryonic development, underscoring their dynamic regulatory importance in this developmental setting [16,[19][20][21][22].
To explain the mechanism of embryonic retardation caused by unknown factors, we attempted to identify some biomarkers that could influence embryonic development by comparing miRNA profiles of normally developing and arrested embryos. Here, we explored the expression of 12 different miRNAs in arrested and developing embryos and ultimately detected miR-145 as an aberrantly upregulated miRNA in arrested samples. We then explored the regulatory role of this miRNA via the microinjection of miR-145 mimics into murine oocytes and monitored the development and quality of the resultant embryos generated via IVF. We further identified Abca1 as a putative miR-145 regulatory target and determined that miR-145 could suppress the protein level expression of ABCA1. Furthermore, we determined that ABCA1 overexpression could rescue mouse embryo arrest induced by miR-145 overexpression. These results suggest that miR-145\ABCA1 plays a significant role in the preimplantation development and could be used as a therapeutic target to improve the embryo quality.

Informed consent
The Ethics Committee of the Suzhou Municipal Hospital confirmed this research (Research License 2,014,004). All embryos were obtained with written informed consent signed by the donor couples. The informed consent confirmed that the couple donors were voluntarily donating embryos for research on human early embryonic development mechanisms with no financial payment (Research License 2,014,004).

Study subjects
From January 2018 to November 2019, 28 couples suffering from obstructive azoospermia (OA) were recruited from The Affiliated Suzhou Hospital of Nanjing Medical University. All of these couples met the following enrollment criteria: 1. Both partners were 20-40 years of age and men were diagnosed with OA; 2. The female partner did not exhibit any apparent abnormalities concerning the ovulatory status or oocyte morphology; 3. Table 1 details the clinical features of the 28 infertile women included in this paper; 4. The couples had undergone oocyte fertilization via ICSI. Spermatozoa (sperm cells) were obtained by microdissection from the testis or epididymis of male partners. All 28 couples have had successful pregnancies and births through ICSI at the center and have been authorized to remove excess embryos from freezing storage.

Collection and culture of human embryos
Each couple donated one or two cryopreserved Day 3 cleavage-stage embryos, a total of 51. The embryos were thawed rapidly by taking straws from the liquid nitrogen storage tank, exposing them to air for 40 s, and immersing them in a water bath at 30 °C for 1 min. Embryos were sequentially placed into thawing medium drops for 5 min with decreasing PROH concentrations (1.0 mol/l, then 0.5 mol/l and finally 0 mol/l), each with 0.2 mol/l sucrose at room temperature to remove the cryoprotectant. Thawed embryos were transferred to PBS with 20% HSA for 10 min at 37 °C and then to G2 culture medium (Vitrolife) to evaluate blastomere survival. The G-2 (Vitrolife) medium was used to culture the 8-cell embryos to the blastocyst stage. At the 6th day, the blastocysts with a thin zona pellucida, smooth trophectoderm, clearly visible blastocyst cavity and well-developed inner cell mass were collected as "Normal Controls". The arrested 8-cell and morulae embryos that have no signs of degeneration were collected as "Arrested embryo". In this study, 15 normal controls and 15 arrested embryos were collected for the miRNA quantification.

Embryonic phenotype analyses
The morphological characteristics of embryos during different stages of development were assessed via light microscopy (IX-71, Olympus, Japan). Embryo quality was assessed at pre-selected time points using standard guidelines [23].

Mice
Wild-type (WT) ICR mice were acquired from the Laboratory Animal Center of Nanjing Medical University, China. They were kept in specific pathogen-free circumstances in a climate-controlled setting (50-70% humidity, 20-22 °C, 12 h light/dark cycle) with free access to water and food. Animal experiments were approved (license number 2004020) by the Animal Ethical and Welfare Committee of Nanjing Medical University (Nanjing, China).

Superovulation and oocyte collection
Female 21-23 days old mice were intraperitoneally injected with 5 IU of pregnant mare's serum gonado¬tropin (PMSG, Ningbo Sansheng Pharmaceutical) to induce superovulation. At 46 h post-injection, mice were euthanized by cervical dislocationThe full grown oocytes (FGO) were collected by manual puncture of ovarian follicles and maintained in M2 medium (Sigma, St. Louis, MO, USA) containing 2 μM milrinone to inhibit GVBD.

Oocyte microinjection
The overexpression of miR-145 and miR-145-mu were attained through purchasing miR-145 mimic\mu from GenePharma and diluting them to a final concentration of 20 uM. To overexpress Abca1, a capped cRNA was synthesized as described below. An Eppendorf Transferman NK2 micromanipulator was utilized to conduct the microinjection of the miRNA mimic\mu,cRNA or distilled water (Control), to denuded oocytes with an injection volume of 5-10 pL. In addition, a group of embryos that had not undergone microinjection served as culture controls (Normal). Once this injection was complete, oocytes were rinsed and cultured in an M16 medium for 14 h.

In vitro fertilization
A moderately revised version of an already reported approach was used to conduct all murine IVF experiments [24]. Sperm were collected from dissected epididymis samples from 10 to 12 week-old ICR mice. Following the incubation of these samples in HTF medium (Millipore, Merck) containing 10 mg/ml BSA for 1 h, dispersed spermatozoa were added to HTF drops with oocytes. Following coincubation at 37 °C in an incubator for 4-6 h, the presumptive zygotes were rinsed and transferred to KSOM media (Millipore). They were cultured to the blastocyst stage at 37 °C in a moisturized incubator under a 5% CO2, 5% O2, and 90% N2 atmosphere.

Cell culture
HEK293T cells were procured from the China Infrastructure of Cell Line Resources and grown in DMEM supplied with 10% fetal bovine serum (FBS; Hyclone, UT, USA) and penicillin/streptomycin. The cells were cultured in a 37 °C 5% CO2 incubator.

Western blotting
Samples were incubated at 95 °C for 5 min in 2 × SDS sample buffer and stored at − 20 °C. For the embryos, a total of 100 samples per group were studied. For 293 T Cells, 5 × 10 6 cells per group were used. Proteins extracts were separated using 10% SDS-PAGE and transfer onto PVDF membranes (Amersham Pharmacia Biotech, Herts, UK). Blots were then proved overnight with monoclonal mouse anti-ABCA1 (Abcam, MA, USA; ab18180) or mouse anti-GAPDH (Abcam, MA, USA; ab8245) at 4 °C. Appropriate HRP-conjugated anti-mouse IgG secondary antibodies were then used for protein detection, followed by an ECL kit (Thermo Scientific ™ , 32,106) was implemented to visualize protein bands. GAPDH was employed as a loading control.

Cholesterol analyses
Total embryonic lipid content was extracted using methanol-chloroform, followed by a cholesterol determination kit (Sigma-Aldrich, MO, USA; MAK043) based on provided directions to measure cholesterol content.

In vitro transcription of cRNA
Murine Abca1 plasmids were purchased from GeneCopoeia. Murine Abca1 was amplified from murine embryonic cDNA and cloned into the pCS2+ vector, which harbors a Myc tag, enabling in vitro polyadenylated mRNA transcription [25]. SacII or KpnI were used for construct linearization, followed by gel extraction kit (Promega, WI, USA; A9281)-mediated purification. Capped mRNAs were generated with an SP6 message machine (Ambion, CA, USA; AM1340) employed for generating capped mRNAs, and a RNeasy cleanup kit (Qiagen, 74,204) was utilized for subsequent mRNA purification.

Analysis of differential miRNA expression patterns in arrested blastocysts
We used donated embryos to detect the expression patterns of 12 different miRNAs.. To exclude obvious maternal factors, we selected patients whose disease was due to male OA, and all examination parameters of females were normal, as described in detail in the materials and methods. Through qPCR, miR-29b, miR-101, miR-200b, miR-145, miR-34a, and miR-25 were found to be upregulated in arrested blastocysts (P < 0.05). At the same time, miR-22 and miR-30c were downregulated relative to normal control embryos (P < 0.05). No differences in miR-26a, miR-374a, miR-21, or miR-128 expression were observed between these groups (Fig. 1). Given that miR-145 exhibited the highest levels of differential expression among the surveyed miRNAs, we hypothesized that it could perform an essential task in the context of embryonic development.

miR-145 overexpression in murine oocytes modulates early embryonic potential
To discover the effective functional task of miR-145 during the early stages of embryogenesis, we used a miR-145 mimic microinjection technique to overexpress it in murine oocytes. These oocytes were then utilized for IVF assays conducted using normal ICR mouse sperm,  (Fig. 2). These data suggest that overexpressing miR-145 can result in the arrest of embryonic development.

miR-145 directly targets Abca1 to regulate its expression
To further illuminate the regulatory roles of miR-145 in the context of embryogenesis, we used the Targetscan database (www. targe tscan. org) to identify putative miR-145 target genes. Revealing Abca1 is one such target gene (Fig. 3A). To confirm the capability of miR-145 for direct regulation of the expression of murine Abca1, we generated a luciferase reporter construct harboring the Abca1 3′-UTR sequence predicted to harbor a miR-145 binding site. A dual-luciferase reporter assessment was then conducted. And the reduction in relative luciferase activity was detected in cells transfected with miR-145 (Fig. 3B), while this effect was abolished when 9 nucleotides within the putative miR-145 binding region were mutated. To clarify whether miR-145 can regulate ABCA1 expression under endogenous conditions, the 293 T cells were transfected with miR-145 mimic or mutant control constructs. The expression of miR-145 and ABCA1 at the protein and mRNA levels were assessed. As expected, ABCA1 mRNA and protein levels were substantially reduced in cells transfected with the miR-145 mimic but not the mutant version of this construct (Fig. 3C-E). It was revealed that Abca1 is a target gene.

miR-145 influences embryonic cholesterol levels via the regulation of ABCA1 protein expression
To fully clarify the functional role of embryonic miR-145, we microinjected murine oocytes with miR-145 or a mutant control construct. These oocytes were then used to prepare blastocysts via IVF, and Abca1 expression was appraised at the protein and mRNA levels through Western blotting and qPCR assays. This approach revealed that ABCA1 was significantly downregulated at the protein and mRNA levels in miR-145 overexpression, whereas the mutant miRNA construct failed to impact such expression ( Fig. 4A-C).
ABCA1 is a crucial regulator of phospholipid and cholesterol homeostasis in cells that are also referred to as CERP (cholesterol efflux regulatory protein). Abnormal ABCA1 expression patterns can lead to the aberrant accumulation or depletion of intracellular cholesterol. We thus assessed cholesterol levels in the embryos in our different treatment groups, revealing a significant increase in cholesterol levels in embryos overexpressing miR-145 (Fig. 4D).

Appropriate miR-145/Abca1 homeostasis is essential for normal embryonic development
To directly evaluate the mechanistic link between miR-145 upregulation and embryonic developmental arrest, we overexpressed Abca1 in oocytes overexpressing miR-145 using the same microinjection technique. We then compared the embryo to blastocyst ratio in each group. This analysis revealed that the overexpression of miR-145 significantly decreased embryo competence, as evidenced by reduced blastocyst development (Fig. 5A, B), consistent with our findings above. However, the overexpression of Abca1 was sufficient to partially rescue such miR-145-mediated inhibition of embryonic development (Fig. 5A, B). As expected, Abca1 mRNA levels were lower in embryos that had been microinjected with miR-145. At the same time, this was reversed in the context of supplemental Abca1 mRNA microinjection (Fig. 5C). In addition, Abca1 microinjection was sufficient to reverse the miR-145-induced increase in cholesterol levels within these embryos (Fig. 5D). Together, these data thus suggest that miR-145 can impair embryonic development. In contrast, the Abca1-mediated restoration of cholesterol homeostasis can restore normal embryogenesis.

Discussion
Many couples affected by infertility experience recurrent IVF/ICSI failure. In some cases, patients may exhibit normal ovulatory status and morphologically normal oocytes yet are nonetheless affected by severely impaired zygote formation and embryonic development. The failed fertilization and embryonic arrest phenotypes observed in these patients are collectively referred to as PEL. Herein, we sought to discover the molecular determinants of such fertility outcomes by collecting normal and arrested embryos from couples undergoing ICSI treatment at our center. When we scrutinized the expression levels of different miRNAs in these embryos via qPCR, we found that miR-145 was overexpressed in the arrested embryos relative to the normally developing embryos, suggesting high levels of expression of this miRNA may be tied to impaired embryonic development. Furthermore, mouse embryo experiments demonstrated that the high level of miR145 inhibited embryonic development, especially the development from 8-cells to blastocyst stage. Therefore, our data suggest that miR145 plays a regulatory role during preimplantation embryonic development. Similar miRNA profiles in both embryos during the early stages of development and mature murine oocytes suggest that zygotes primarily contain maternally-derived miRNAs [26]. The expression of these miRNAs is reduced by up to 60% between the 1and 2-cell stages of development but doubles at the end of the 4-cell stage relative to the 2-cell stage, consistent with embryonic genomic activation initiating somewhere between the 1-and 4-cell stages of embryogenesis [26]. While miRNAs are routinely synthesized and degraded throughout murine preimplantation embryo development, an overall rise in miRNA expression levels is observed towards the blastocyst stage [27]. While many studies have examined miRNA expression dynamics in mice, the specific functional tasks of individual miRNAs in the context of early embryonic development remain poorly understood [28,29].
miR-145 has been reported to repress the expression of genes associated with pluripotency in human ES cells in prior studies [30]. And treatment of nuclear-transferred embryos with a miR-145 inhibitor improves developmental competence and quality. miR-145 was chosen as a target for additional research. Other research suggests that miR-145 can target a range of oncogenes, thereby modulating cellular proliferative, migratory, invasive, and apoptotic activity [31]. When we employed a microinjection approach to overexpress miR-145 in murine oocytes, we found that such overexpression significantly increased The overexpression of miR-145 in 293 T cells was confirmed via qPCR. D, E Abca1 mRNA and protein levels (cropped gels/blots) were assessed by means of qPCR and Western blotting after the indicated treatments. All the data are means ± s.d. from five independent experiments the rate of arrest during embryogenesis, suggesting that high levels of miR-145 expression can trigger arrest during embryonic development.
ABCA1 is an ABC1 superfamily member responsible for transporting cholesterol and phospholipids across the cell membrane to HDL-C, thus playing a key role in regulating cholesterol homeostasis, endothelial function, blood pressure regulation, vascular inflammation, and platelet aggregation in the context of atherosclerotic vascular disease [32]. Several miRNAs have been reported to regulate Abca1 expression, including miR-33a, miR-122, miR-467b, miR-183, and miR-28 [33,34]. miR-145 has been found to promote hypoxia-induced proliferation and migration of pulmonary arterial smooth muscle cells by regulating ABCA1 expression [35]. However, so far, no studies have investigated the function of ABCA1 during preimplantation embryo development. Here, we verified that Abca1 is a straightforward miR-145 target in mice embryos, with miR-145 overexpression consequently resulting in significant decrease in ABCA1 levels within embryos, potentially contributing to the consequent incidence of developmental arrest during embryogenesis.
Cholesterol has been found to be essential for preimplantation embryo development, and cholesterol depletion inhibited preimplantation development [36]. Cholesterol is one of the main components of lipid rafts. Mouse preimplantation embryos express stagedependent distribution of lipid rafts, which possibly play crucial roles in cytokinesis and cell signaling events at this stage by acting as a signal organizing platform [36]. In addition, Pawlak et al. identified significant involvement of cholesterol metabolism in preimplantation embryos by RNA sequencing of porcine and bovine preimplantation embryos [37]. In our study, we found that Abca1 overexpression in this experimental system was sufficient to reverse the miR-145-induced arrest of embryogenesis through mechanisms tied to reductions in cholesterol content within embryos. This